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Receptor tyrosine kinase signaling pathway analysis for diagnosis and therapy

a receptor tyrosine kinase and signaling pathway technology, applied in the field of receptor tyrosine kinase signaling pathway analysis for diagnosis and therapy, can solve the problems of lack of sensitivity, difficult application of techniques, and difficulty in studying the signaling pathway, and achieve the effect of overcompensating deficiencies in the ar

Active Publication Date: 2008-07-22
MONOGRAM BIOSCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Signaling pathways have been difficult to study not only because of their complexity and interconnectedness, but also because of the disruptive procedures required for analysis of intracellular complexes, e.g. Weng et al, Science, 284: 92-96 (1999); Machida et al, Molecular & Cellular Proteomics, 2.4: 215-233 (2003); Price et al, Methods in Molecular Biology, 218: 255-267 (2003).
Unfortunately, such techniques are difficult to apply, generally lack sufficient sensitivity to provide an accurate picture of the state of a signaling pathway, and / or cannot measure multiple components or interacting components that are crucial for pathway activation.

Method used

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  • Receptor tyrosine kinase signaling pathway analysis for diagnosis and therapy
  • Receptor tyrosine kinase signaling pathway analysis for diagnosis and therapy
  • Receptor tyrosine kinase signaling pathway analysis for diagnosis and therapy

Examples

Experimental program
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Effect test

example 1

Simultaneous Measurement of Her2-Her3 Heterodimerization and Erk1 Phosphorylation

[0143]In this example, an assay is described for providing a ratiometric measure of phosphorylated Erk1 and Her2-Her3 heterodimerization. The assays are carried out as follows.

Sample Preparation:

[0144]1. Serum-starve breast cancer cell line culture (MCF-7) overnight before use.[0145]2. Stimulate cell lines with HRG in culture media for 10 minutes at 37° C. Exemplary doses of HRG are 0, 0.032, 0.16, 0.8, 4, 20, 100 nM for MCF-7 cells.[0146]3. Aspirate culture media, transfer onto ice, and add lysis buffer (described below) to lyse cells in situ.[0147]4. Scrape and transfer lysate to microfuge tube. Incubate on ice for 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min.[0148]5. Collect supernatants as lysates and aliquot for storage at −80° C. until use.

Lysis Buffer (Made Fresh and Stored on Ice):

[0149]

FinalulStock1% Triton X-100100010% 20 mM Tris-HCl (pH 7.5) 200  1 M100 mM NaCl 200  5 M 50 mM NaF 500  1...

example 2

Analysis of Cell Lysates for Her-2 Heterodimerization and Receptor Phosphorylation

[0167]In this example, Her1-Her2 and Her2-Her3 heterodimers and phosphorylation states are measured in cell lysates from several cell lines after treatment with various concentrations of epidermal growth factor (EGF) and heregulin (HRG). Measurements are made using three binding compounds and a cleaving probe as described below.

Sample Preparation:

[0168]1. Serum-starve breast cancer cell line culture overnight before use.[0169]2. Stimulate cell lines with EGF and / or HRG in culture media for 10 minutes at 37° C. Exemplary doses of EGF / HRG are 0, 0.032, 0.16, 0.8, 4, 20, 100 nM for all cell lines (e.g. MCF-7, T47D, SKBR-3) except BT20 for which the maximal dose is increased to 500 nM because saturation is not achieved with 100 nM EGF.[0170]3. Aspirate culture media, transfer onto ice, and add lysis buffer to lyse cells in situ.[0171]4. Scrape and transfer lysate to microfuge tube. Incubate on ice for 30 m...

example 3

Analysis of Tissue Lysates for Her2 Heterodimerization and Receptor Phosphorylation

[0193]In this example, Her1-Her2 and Her2-Her3 heterodimers and phosphorylation states are measured in tissue lysates from human breast cancer specimens.

Sample Preparation:

[0194]1. Snap frozen tissues are mechanically disrupted at the frozen state by cutting.[0195]2. Transfer tissues to microfuge tube and add 3×tissue volumes of lysis buffer (from appendix I) followed by vortexing to disperse tissues in buffer.[0196]3. Incubate on ice for 30 min with intermittent vortexing to mix.[0197]4. Centrifuge at 14,000 rpm, 4° C., for 20 min.[0198]5. Collect supernatants as lysates and determine total protein concentration with BCA assay (Pierce) using a small aliquot.[0199]6. Aliquot the rest for storage at −80° C. until use.

Assay Design:[0200]1. The total assay volume is 40 ul.[0201]2. The lysates are tested in serial titration series of 40, 20, 10, 5, 2.5, 1.25, 0.63, 0.31 ug total-equivalents and the volume...

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Abstract

The invention provides a method for determining the activation status of receptor tyrosine kinase (RTK) pathways in either cell samples or patient samples by measuring receptor dimerization and relative amounts of protein-protein complexes or activated effector proteins that are characteristic of an RTK pathway. The invention also provides a method of using such status information to select patients responsive to pathway-specific drugs, and more particularly, to methods for measuring ErbB receptors and receptor complexes and using such information to select patients responsive to ErbB pathway-specific drugs. Preferably, methods of the invention are implemented by using sets of binding compounds having releasable molecular tags that are specific for multiple components of one or more complexes formed in RTK activation. After binding, molecular tags are released and separated from the assay mixture for analysis.

Description

[0001]This application claims priority from U.S. provisional applications Ser. No. 60 / 510,910 filed 14 Oct. 2003; Ser. No. 60 / 566,352 filed 28 Apr. 2004; Ser. No. 60 / 571,816 filed 17 May 2004; and Ser. No. 60 / 577,256 filed 03 Jun. 2004; which applications are incorporated by reference in their entireties.FIELD OF THE INVENTION [0002]The present invention relates generally to methods for determining the status of receptor tyrosine kinase (RTK) signal transduction pathway activation and for using such status information to select patients responsive to pathway-specific drugs, and more particularly, to methods for measuring ErbB receptors and receptor complexes and using such information to select patients responsive to ErbB pathway-specific drugs.BACKGROUND OF THE INVENTION [0003]Signaling pathways between the extracellular environment and the nucleus of a cell involve the formation of many molecular complexes in which multiple proteins are assembled to directly or indirectly induce m...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): G01N33/53G01N33/533A61BA61K31/4745C12Q1/68G01N33/50G01N33/574G01N33/68G01N33/74
CPCG01N33/5041G01N33/57492G01N33/74G01N33/6893G01N2333/485
Inventor MUKHERJEEI, ALITANG, MENGXIANGPANNU, HARPRIT S.CHAN-HUI, PO-YINGSINGH, SHARAT
Owner MONOGRAM BIOSCIENCES
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